Porcine adipocyte antigens and their use in the immunological control of fat

ABSTRACT

Antigens present in the plasma membrane of mature porcine white adipocytes, which are not present in porcine liver, kidney, spleen, brain, cardiac muscle, skeletal muscle or lung or in porcine erythrocytes, which react with antisera raised against said adipocytes and which on SDS-PAGE give rise to protein bands of relative molecular mass (r.m.m.) about 37, 50, 51 and 121 KiloDaltons, respectively and antibodies thereto are useful for the reduction of fat in pigs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the immunological control of fat in themammalian body, especially in non-human animals (herein referred tosimply as animals).

2. Description of Prior Art

Excess fat in animals is recognised as detrimental to loweringproduction costs and a health risk to human consumers. One attempt atreducing the deposition of fat in animals has been to incorporate a5-agonist in the feed. 5-agonists have encountered many problems,particularly that they can adversely affect meat quality and itspreservation during storage and that the animals have to be slaughteredwithin a short period after the compound is withdrawn from the feed.Another attempt has been the use of growth hormones such as bovinesomatotrophin (BST). Besides stimulating milk yield, BST improves theprotein: fat ratio and feed conversion efficiency in cattle. Althoughthe dairy industry considers BST to be safe, it has been the subject ofconsiderable concern to regulatory authorities and consumer groups.

In view of these problems, D J Flint et al., International Journal ofObesity 1, 69-77 (1986) pioneered the idea of raising antibodies to theplasma membranes of adipocytes and injecting them into animals. It wasfound that this treatment reduced the amount of fat considerably andthat this reduction was maintained for several months without inducingadverse effects. The first published reports, in which crude antiseraraised against whole adipocyte plasma membranes from rats were shown tohave such an effect, were by D J Flint, H Coggrave, C E Futter, M JGardner and T Clarke, International Journal of Obesity 1, 69-77 (1986)and by D J Flint and C E Futter, Annual Report of the Hannah ResearchInstitute, Ayr, Scotland 1986. Not only was fat reduced, that there wasa body weight gain and an improvement in feed conversion efficiency. Inan article "Can obesity be controlled?" by D J Flint, C E Futter and MPeaker, News in Physiological Sciences 2, 1-2 (February 1987), it isreported that similar antibodies have been produced against sheep andpig fat cells and that all are effective against adipocytes in vivo. Amore detailed report on the effects of treatment of Fats withanti-adipocyte antibodies is given by D Panton, C E Futter, S Kestin andD J Flint in American Journal of Physiology 258, (Endocrinol. Metab.21): E985-E989 (1990). See also A P Moloney and P Allen, Proc. NutritionSociety, July 1988 Meeting, page 14. Although J Killefer and C Y Hu,Proc. Soc. Exp. Biol. Med. 194, 172-176 (1990) have reported raising amonoclonal antibody to pig adipocyte plasma membranes, the hybridoma isbelieved not to be publicly available and the paper contains no evidencethat the antibody would lyse fat cells. J T Wright and G J Hausman, Int.J. Obesity 14, 395-409 (1990) report the preparation of monoclonalantibodies against porcine adipocyte plasma membranes of 2-week oldpigs. The antibodies are reported to immuno-precipitate proteins ofrelative molecular mass 77 and 90 kD. These experiments were directed toidentifying cell surface antigens useful as markers of differentiatingadipocytes.

J. Killefer and C Y Hu, J. Cell. Biochem. 44, 167-175 (1990) describe a64 kD protein present in the plasma membrane of adipocytes andgenetically lean pigs, but not in adipocytes of genetically obese pigs.

Although some of the above work has demonstrated experimentally thepossibility of treating fat deposition in vivo by the administration ofanti-adipocyte antibodies, it is a problem that the production of suchantibodies may be very labour-intensive. The administration of theplasma membranes themselves as antigens could be considered, if theycould be conjugated to carrier proteins and could thereby by made"non-self". However, the production of plasma membrane material fromslaughterhouses poses difficulty of quality control. If the antigen(s)responsible for the fat reduction could be isolated and purified, theway would be open to making them by a recombinant DNA method or byprotein synthesis.

SUMMARY OF THE INVENTION

After considerable research, the inventors have isolated from porcinefat cell plasma membranes, antigens which appear to be specific toadipocytes (at least in the sense of not being detectable in many otherbody tissues of the animal) and reactive with antibodies to fat cellplasma membranes. However, not all of these antigens give rise toantisera which do, in fact, reduce fat cell deposition in vivo. Aftertests of sheep anti-porcine fat cells plasma membranes in vivo on pigs,the inventors have found certain antigens which can produce suchantibodies. Accordingly, the present invention provides 4 antigenspresent in the plasma membrane of mature porcine white adipocytes, whichare not present in porcine liver, kidney, spleen, brain, cardiac muscle,skeletal muscle or lung or in porcine erythrocytes, which react withantisera raised against said adipocytes and which on SDS-PAGE give riseto protein bands of relative molecular mass (r.m.m.) about 37 (A), 50(B), 51 (C) and 121 (D) KiloDaltons, respectively, as determined bymarkers of relative molecular mass 29, 45, 66, 97, 116 and 205KiloDaltons. These values for proteins A, B, C and D are expressed asthe nearest whole number and are subject to possible error of up toabout 2.5%.

12 candidate antigens designated 1a, 1b, 2-6, A-F and H, all potentiallyfat cell specific, were resolved by SDS-PAGE, with difficulty. The onlyway in which It could be determined whether they would reduce fatdeposition was to raise antibodies and test them in pigs. As a result ofthese tests it was determined that A, B, C and D, at least, are active.

The invention also provides the use of such antigens and antibodiesthereto for the active or passive immunisation of sheep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a Coomassie blue stained gel of porcine adipocyte plasmamembrane polypeptides separated by SDS-PAGE with a laser densitometryscan profile, showing the positions of the candidate antigens;

FIG. 2 is a photograph of an immunoblot showing the interaction of anantiserum to adipocyte plasma membranes with protein bands obtained fromvarious porcine tissues including adipocyte plasma membranes;

FIG. 3 shows ELISA data for reactivity to whole adipocyte plasmamembranes of various antisera raised against the candidate antigens;

FIG. 4 (a) and (b) show sections through the backfat of pigs treated (a)with control antiserum and (b) with antibodies to whole adipocyte plasmamembranes, illustrating the depletion of fat which occurs afteradipocyte destruction; and

FIG. 5 (a), (b), (c) and (d) are photographs of typical histologicalsections of back fat treated with control serum (d) and antibodies ofthe invention (a, b, c) respectively.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The adipocyte membranes used to prepare the antigens can be obtainedfrom any porcine breed as it is likely that they will be highlyconserved between breeds. It is suggested that they be obtained frommature white adipocytes. A mature adipocyte is one which demonstratesmorphologically and biochemically the adipocyte phenotype including thepresence within the cell of a large central unilocular lipid droplet.All references to "adipocytes" herein mean mature adipocytes. It is alsosuggested that the adipocytes be obtained by a gentle procedure whichdoes not damage them and which removes extracellular material. It hasbeen found that a Clostridium histolyticum collagenase enzyme isparticularly useful for such removal. The cells are then allowed torecover by a conditioning step of incubation at a temperature of 37° C.for a period of several hours, e.g. 2 to 24 hours.

The adipocytes can then be separated and washed by flotation, retainingthe floating adipocyte layer, which is then homogenised and the plasmamembranes separated on a discontinuous sucrose gradient. It is thennecessary either to remove non adipocyte-specific antigens or toidentify which of the antigens present in the extract areadipocyte-specific. For this purpose, other porcine tissue is required.The greater the number of Kinds of tissue employed for this purpose, thebetter the chances will be of obtaining adipocyte-specific antigens. Theinventors have found it preferable to make a direct protein bandimmunoblot comparison by running plasma membrane extracts prepared fromvarious porcine tissues on SDS-PAGE, Western blotting and thenimmunoblotting specific protein bands with antibodies raised againstplasma membranes of adipocytes. This immunodetection was carried outwith difficulty, by scanning laser densitometry. Comparing the laserscans from the adipocyte plasma membranes with those from other tissues,12 candidate "adipocyte specific antigens" were identified from theblots of a large number of proteins.

In order to generate sufficient material by this method, large scalepreparations were carried out on SDS-PAGE and the bands containing eachof the 12 antigens were cut from the gels and proteins were thenelectroeluted therefrom. Antisera were raised against the 12electroeluted products and 10 of these were found to reactimmunogenically with whole adipocyte plasma membranes. The antisera werethen tested in vivo by passive immunisation of pigs by local injectionsinto areas of the body having significant subcutaneous fat. The largestlesions, indicating the reduction of fat, resulting from this treatmentwere those induced by anti-whole adipocyte plasma membranes,anti-antigen A and anti-antigen C, although some useful results wereobtained from antigens B and D. The invention therefore includes theseantigens. D is currently thought to have the weakest effect.

The information given herein enables antigens to be identified, isolatedand, by methods well known in the art, purified. It is then possible toprepare antibodies by conventional raising of antisera or production ofmonoclonal antibodies by the Kohler-Milstein method and variationsthereon. Immunogenic portions of natural antibodies are also includedwithin the scope of the term "antibodies" as used herein, as are alsohybrid human-mouse antibodies and other antibody-like productsobtainable by recombinant DNA methods.

The antibodies thereby produced will be used to help identify antigensfrom the adipocyte membrane by a recombinant DNA method. The preparationof such antigens was begun by producing a cDNA library from RNA obtainedfrom isolated adipocytes using a guanidinium isothiocyanate extractionmethod (Chomczynski and Sacchi, Analyt. Biochem. 162, 156-159 [1987])followed by affinity chromatography to enrich for poly A+ RNA (Avid andLeder, Proc. Natl. Acad. Sci. (USA) 69, 1408-1412 [1972]). The cDNAprepared from this enriched RNA was inserted into the phage expressionvector λgt 11. The correct clones will be identified by using either theantibodies previously mentioned (Mienendorf et al., Methods inEnzymology 152, 451-457 [1987]) or by synthesising a mixture of the mostprobably complementary oligonucleotides, as determined from amino acidsequence of the antigens and considerations of normal DNA codon usage,as probes. The clones thus identified and isolated, after confirmingtheir binding of antibody, can be used to express the protein ofinterest.

The main use of the invention will be for the treatment of excess fatproduction In pigs. While either active or passive immunisation islikely to have an effect, active immunisation is preferred and for thispurpose the antigen will have to be made "non-self" so that it does notsuffer host immune tolerance. This can be achieved by any of theconventionally explored methods, especially by conjugation to a carrierprotein such as rabbit serum albumin or KLH. Epitopes of the antigen canbe identified thereby enabling shorter-chain peptides to be used asimmunogens. The Invention includes these within its scope. Such peptidescan also be used In the form of conjugates or other elaboratedstructures such as branched forms thereof, in which the peptide ispresented on "arms" of a carrier such as a branched lysine core.

The favoured proposed route of administration for active immunisation isby subcutaneous injection. Amounts of antigen in the range of 1 μg to 1g per treatment are envisaged, the animal being treated preferably onceonly for reasons of convenience of husbandry. Adjuvants such as smallcomponents of Freund's Complete Adjuvant or peptides can be employed.Oral or nasal routes are possible alternatives.

For passive administration, the antibodies are preferably given withoutan adjuvant, again preferably by subcutaneous injection. Other routessuch as intraperitoneal or oral are possible.

The following Examples illustrate the invention.

EXAMPLE Section 1

Isolation of mature white adipocytes from porcine adipose tissue

Methods

Adipose tissue was collected from the following depots of freshly killedpigs: channel, mesenteric, perirenal and subcutaneous. The mixed adiposetissue was finely minced and collagenase at 1 mg/ml in medium 199 (GibcoBiocult) and 1% normal pig serum was added (20 ml of medium to 10 gtissue). The tissue was shaken at 37° C. for 1.5 hours and isolatedadipocytes obtained by straining through a sieve. The adipocytes werewashed three times by flotation and then conditioned for 2 hours inmedium 199 containing 1% normal pig serum at 37° C. The isolatedadipocytes were stored at -20° C. until used.

The cells isolated in this manner have been shown to be greater than 90%Intact and viable using standard cell viability tests, and maximalimmunoreactivity had been achieved.

Section 2

The isolation and preliminary characterisation of plasma membranepreparations from conditioned porcine adipocytes

Methods

Adipocytes, isolated and conditioned as described in Section 1, wereused for the preparation of plasma membranes. Typically, 30 ml ofadipocytes were mixed with 30 ml of sucrose-based extraction medium(0.25M sucrose, 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM PMSFphenylmethyl sulphonyl fluoride) prewarmed to 37° C. The suspended cellswere then disrupted by mixing for 3×30 sec on a vortex mixer. The finalhomogenate was centrifuged at 1000 g.av. for 5 min. Followingcentrifugation, the material beneath the floating plug of fat wasremoved by aspiration and kept on ice. This was then centrifuged at100,000 g for 1 hour. The supernatant was then removed and the pelletresuspended in 2 ml of 50 mM Tris buffer using a plastic Pasteurpipette. Discontinuous sucrose density gradient centrifugation was thenused to separate mitochondria and other components from the plasmamembranes. The membranes were removed from the 0/32% sucrose interfaceand pelleted at 100,000 g for 1 hour at 4° C. The pellet was resuspendedin 50 mM Tris HCl, pH 7.4 and stored at -20° C. until used.

Section 3

The replicated SDS-PAGE analysis of the pattern of polypeptides fromporcine adipocyte plasma membranes

Methods

Preparation and running of analytical gels

The above-prepared plasma membranes from porcine adipocytes wereanalysed by SDS-PAGE using a linear gradient of polyacrylamideconcentration between 5 and 15% for the resolving gel. The gels wereprepared and run by a modification of the procedure originally outlinedby Laemmli, Nature 227, 680-685 (1970) in a vertical electrophoresisunit (Atto Corporation) according to the manufacturer's instructions.The electrophoresis was performed in 7 by 8 cm "minigels" (0.75 mmthick). See also Tume, Lee and Cryer, Comp. Biochem. Physiol. 80B,127-134 (1985).

Protein standards (Biorad high molecular weight 205, 116, 97, 66, 45 and29 KiloDaltons were heated to 37° C. for 2 hours in the presence of anequal volume of loading buffer. [1.0 ml stacking gel buffer, 0.125MTris-HCl, pH 6.8, containing 0.1% (w/v) SDS, 400 μl 10% glycerol, 200 μl2-mercaptoethanol, 100 μl 0.25% bromophenol blue, 300 μl distilledwater]. Pig adipocyte plasma membranes were treated in similar fashionfor 30 min. Electrophoresis was performed at 150 V constant voltage. Theelectrode buffer used was 0.25M Tris containing 0.192M glycine and 0.1%(w/v) SDS, at pH 8.3.

Staining and destaining of gels

Gels were immersed for 1-2 hours in 0.125% (w/v) Coomassie BrilliantBlue in 10% (v/v) glacial acetic acid containing 40% methanol. Tovisualise protein bands, the gels were then destained in repeatedchanges of 10% (v/v) acetic acid in 30% (v/v) methanol.

Calibration using Standards of Known relative molecular mass

The relationship between relative molecular mass and mobility wasdetermined by loading analytical gels with a standard mixture ofprestained polypeptides of known relative molecular mass (Biorad highmolecular weight) as recited above and in the "Summary of theinvention". The relative mobility of each polypeptide was calculated bymanual measurement of the gels and plotted against log.₁₀ molecularmass. The gradient, calculated by regression analysis, was found to belinear, allowing molecular weights of unknowns to be determined byinterpolation.

Laser scanning densitometry

The gels, stained and destained to reveal protein bands, were scanned bya laser scanning densitometer, whereby the intensity (optical density)of the bands is converted into a peak height and their size isrepresented by the area under the peak. Table 1 shows the relativemolecular mass of the specific antigens and the relative amounts ofprotein represented by areas under peaks determined from the calibrationplot. In FIG. 1 of the drawings, the SDS-PAGE bands were matched to thelaser densitometric scan.

                  TABLE 1                                                         ______________________________________                                        Molecular weights and relative abundance of adipocyte                         antigens used for immunisation of sheep                                               Molecular wt                                                                              Abundance                                                 Antigen (Daltons)   (% of total membrane protein)                             ______________________________________                                        1a      189019 ± 7345                                                                          3.05                                                      b       178395 ± 8130                                                      2       127787 ± 3015                                                                          1.23                                                      3       45488 ± 238                                                                            5.58                                                      4       34729 ± 367                                                                            3.71                                                      5       28014 ± 263                                                                            6.37                                                      6       13608 ± 258                                                                            7.71                                                      A       36949 ± 762                                                                            1.12                                                      B       49553 ± 600                                                                            2.24                                                      C        51244 ± 1123                                                                          4.07                                                      D       121130 ± 2363                                                                          0.93                                                      E       148368 ± 1761                                                                          1.21                                                      F       162055 ± 1831                                                                          0.94                                                      H       79900       N.D.                                                      ______________________________________                                         N.D. Not determined.                                                     

The standard errors for A, B, C and D were 2.0, 1.2, 2.2 and 2.0%respectively.

Section 4

Comparison of the pattern of reactive bands Seen with adipocyte plasmamembranes with similarly treated membranes prepared from other porcinetissues and the use of such comparisons to identify adipocyte specificantigens of the adipocyte membrane

Methods

Preparation of porcine plasma membranes from tissues other than adipose

Collection of tissues

Tissues, other than adipose, were excised from freshly killed pigs. Thesamples were cut Into small pieces (2 g approx) and immersed In liquidnitrogen until completely frozen. The material was then stored at -70°C. until used.

Blood, for the preparation of red blood cell (rbc) membranes, wascollected from the jugular vein of live pigs into heparinised tubes.

Preparation of tissue plasma membranes

10 g of each tissue was finely minced, then 20 ml of membrane extractionmedium (MEM) consisting of 1.4 g Na₂ HPO₄, 81.5 g sucrose, 0.83 g EDTAper liter and 20 μl of PMSF solution (0.2M stock solution) was added.This sample was then homogenised using a tissue homogeniser (3×10 secondbursts), and the resulting homogenate centrifuged at 20,000 g for 20 minto remove dense particulate matter. The resulting supernatant was thencentrifuged at 100,000 g for 1 hour In a Sorvall Combi ultracentrifuge.The resultant pellet was resuspended in 50 mM Tris/HCl.

Sucrose gradients were prepared in polypropylene tubes by overlayingsolutions of 32, 36 and 40% sucrose. The pellets, suspended in Tris/HClwere then applied to the top of the gradient using a Pasteur pipette.The prepared gradient was then spun at 100,000 g for 1 hour.

Following centrifugation, a diffuse band of material was visible at the0/32% interface. This was removed carefully by aspiration and washed inTris and resuspended at an appropriate concentration.

Preparation of porcine red blood cell ghosts

Porcine red blood cell ghosts (i.e. de-haemoglobinised) were preparedusing the method of Raval and Allan, Biochem. Biophys. Acta 856, 595-601(1986). For this, 60 ml of fresh blood was collected into 10 mlheparinised tubes and spun at 2010 g. av. for 10 min. The plasma wasthen removed by aspiration and the remaining packed red cells washedthree times by addition of 10 ml of isotonic Tris/HCl buffer (50 mMTris, 150 mM NaCl, 4.2 mM HCl, adjusted with 1M NaOH to pH 7.4) followedby centrifugation at 2010 g. av. for 10 min. The cells were then lysedby the addition of 10 ml of hypotonic Tris/HCl buffer (50 mM Tris, 4.2mM HCl, adjusted with 1M NaOH to pH 7.4). The cells suspended in thissolution were left at room temperature for 10 min and then the mixturewas centrifuged at 38720 g. av. for 10 min. The sedimented material waswashed 3 times in the lysis buffer. Following this procedure, themembranes, when collected as a pellet, were pale pink to white incolour, with little residual haemoglobin present.

SDS-PAGE immunoblot analysis

FIG. 2 shows the pattern of bands reactive with sheep antibodies toporcine whole adipocyte plasma membranes in various porcine tissueplasma membranes indicating the high degree of reactivity with adiposetissue and the comparatively low reactivity with all other tissue typestested.

Laser densitometry of immunoblots of SDS-PAGE gels

First, (a) blots of SDS-PAGE gels that had been stained with Coomassiebrilliant blue and (b) immunoblots of the same gels against sheepanti-porcine adipocyte plasma membranes, stained with4-chloro-1-naphthol, were scanned using an enhanced laser densitometer(Ultroscan XL LKB). Data collected by the laser densitometer wasanalysed and stored using the Gelscan XL software package. This allowedcomparisons to be made of the polypeptide composition of membranesresolved on SDS-PAGE and of immunoblots of membrane polypeptides (seebelow).

Sheep anti-porcine adipocyte plasma membrane was found to interact withplasma membranes prepared from other porcine tissues, although to asuprisingly low degree. Therefore, laser densitometric scanning, of thereactive bands in adipocyte plasma membranes and in membranes from othertissues, was used to identify adipocyte-specific antigens. That is,immunoblots from the gels of whole adipocyte plasma membranes werecompared with immunoblots from the gels of membrane from other tissues,viz porcine liver, kidney, spleen, brain, muscle, lung and erythrocytes(red blood ghosts).

Identifying adipocyte-specific immunoreactive plasma proteins

The patterns/scans of reactive proteins from adipocyte plasma membraneswere different from the patterns of the membrane proteins from otherporcine tissues and which were themselves different from each other.However, closer comparison showed that some of the immuno-reactiveproteins had similar mobilities although some appeared at specificdifferent intensities of staining, related to the membrane type inquestion. To identify accurately those proteins that were definitelypresent only in adipocyte membranes on the blot, the Rf values of allthe detected proteins in the different tissues were compared with the Rfvalues of the proteins from adipocytes. If any of the other tissues onthe blot had proteins within 0.003 Rf units of any adipocyte membraneprotein, then that protein was assumed to be not specific to adipocytemembranes.

This Rf comparison was performed for each blot separately, then the Rfvalues of amido black-stained molecular weight markers from each blotwere used to plot calibration curves of molecular weight against Rf onlogarithmic paper. These calibration curves were used to find themolecular weights of these initially identified adipocyte specificimmunoreactive membrane components. The relative molecular masses of thespecific adipocyte membrane proteins determined using separate blotswere listed and compared. Those identified on four or more blots wereconsidered to be reproducibly detectable adipocyte specificimmunoreactive membrane components worthy of further investigation.

Results

12 adipocyte-specific antigens were thus found, having the relativemolecular masses shown in Table 1 above. The number of determinationsmade was between 4 and 7 for each antigen. Biorad low molecular weightmarkers were used.

Section 5

Preparation of the above-identified antigens on a larger scale

14×12 cm gels (1.5 mm thick) were used to resolve larger samples ofporcine adipocyte plasma membranes. The buffers and gel solutions usedwere as for the analytical gels. The gels were prepared and run in thesame way, but in a larger electrophoresis unit. They were stained withCoomassie blue as described in Section 3. The procedure was found to bereproducible.

The bands from which polypeptides (A-F, H and 1-6) were to be elutedwere excised from the gels and placed in the sample wells of anelectrophoretic concentrator (Biorad mini-elutor). The proteins wereelectroeluted in a buffer of 25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3 onto a 12,500 molecular weight cut off membrane. Elutions were runat a constant 100 V for 2-3 hours. Eluted samples were removed using apasteur pipette.

Section 6

Removal of SDS and renaturation of polypeptides by dialysis against "OAESephadex" and non-ionic detergent

Removal of the high salt concentration present in the protein samplesafter elution and exchange of SDS for a non-ionic non-denaturingdetergent "Nonidet P40" was achieved by dialysis using a modification ofthe procedure of Hjertan, Biochem. Biophys. Acta 736, 130-136 (1983).

After elution as described above, the samples were placed in 6.3 mmdialysis tubing that had been boiled twice for 10 min in watercontaining 1 mM EDTA. They were then dialysed against 5 liters of 0.2 mM"Nonidet P40", containing 2 g of the anion exchanger "QAE Sephadex", toimprove the efficiency of SDS removal. Dialysis was carried out for 25hours at room temperature with the dialysate being changed after 12hours.

Following dialysis, the samples were stored at -20° C.

Section 7

The use of the individual electroeluted membrane components asimmunogens

Eluted proteins were mixed 1:2 with Freund's complete adjuvant for the1st immunization and subsequently with Freund's incomplete adjuvant.Immunizations were given subcutaneously in 3 sites, 2 in the rump and 1in the shoulder 4 weeks after the initial immunisation. Subsequentimmunisations were carried out at 2 weekly intervals.

Section 8

The demonstration of antibody response using ELISA when each of the 12individual antigens were administered as immunogens

Serum samples collected from sheep injected with each antigen as inSection 7 were tested for their immunoreactivity with adipocyte plasmamembrane (ELISA) or electrophoresed samples of individual antigens orwhole adipocyte plasma membranes electroblotted onto Immobilon(immunoblotting).

ELISA

This was carried out according to the method of Flint et al. (1986).Briefly, porcine adipocyte plasma membranes (1 μg/well in PBS pH 7.4)were coated on to 96 well plates by incubating at 4° C. overnight anddonkey anti-sheep IgG conjugated with horseradish peroxidase was used todetect antibody binding from the antisera raised in sheep againstelectroeluted antigen samples. Samples arising from sheep after thetertiary injection of immunogen (2nd bleed) were tested against ovineadipocyte plasma membranes. Antiserum collected from a sheep previouslytreated with porcine whole adipocyte plasma membranes was used as apositive control. The antisera raised against whole porcine adipocyteplasma membranes reacted strongly with the membranes immobilised on theplate, as did a number of the antisera against specific antigens. FIG. 3(a) to (d) shows plots of four sets of ELISAs in which change in opticaldensity per hour is plotted against the log.₁₀ of the dilution of theantiserum raised against each of the 12 specific antigens and againstwhole porcine adipocyte plasma membranes. It will be seen that antigen 6did not react at all and F scarcely. These two were eliminated fromfurther testing.

Section 9

Establishment of in vivo backfat test in pigs

The rapid testing of antisera for cytotoxicity to adipocytes in vivo

The 10 antisera raised against specific antigens and which gave apositive reaction in the ELISA were tested for their cytotoxicitytowards Intact porcine adipocytes. The test involved the measurement ofthe very obvious lesion that develops around the site of injection of acytotoxic antiserum. These lesions were first observed when pigs werepassively immunised subcutaneously with an antiserum raised in sheepagainst whole porcine adipocyte plasma membranes. Briefly, afterinjection of the antiserum directly into a subcutaneous fat depot, areasof necrotic adipose tissue develop. One week after injection these siteswill appear as areas of necrotic fat and inflamed tissue. Later thenecrotic tissue degenerates, and after 8 weeks the site appears normal,but is devoid of fat, see FIG. 4 (b). No such lesion develops around thesite of Injection of a control antiserum, FIG. 4 (a).

Pigs aged between 6 and 8 weeks were maintained on a standard pigstarter diet. Injection sites were then marked with marker pen on theskin. These sites, four per side, were spaced 5 to 10 cm from the midline and 10 cm apart. Areas known to be relatively devoid ofsubcutaneous fat were avoided.

The antisera from sheep immunised with individual porcine adipocyteplasma membrane antigens, positive control antisera from sheep immunisedwith whole pig adipocyte plasma membranes and negative control sera fromsheep which had received no immunisation were prepared in an identicalfashion, and stored at -20° C. Prior to injection, the antisera werethawed and loaded into individual syringes and warmed to roomtemperature. A mixture of equal proportions of the individual testantisera was also prepared (excluding the + and - control antisera) forcomparison with the antisera to the whole membrane.

Each pig used received one injection of each of the positive andnegative control antisera. In the remaining 6 sites, test antisera wereallocated randomly. Each immunisation was of 1 ml, injected into thesubcutaneous fat.

7 days after treatment, pigs were slaughtered by captive bolt, suspendedby the hind legs and exsanguinated. After all blood flow had ceased, arectangular strip of skin 40 cm wide and including all the injectionsites and surrounding tissue was carefully peeled back by skinning downfrom the rump. This was left attached to the carcass at the neck (toavoid confusing the sites). Care was taken to remove the subcutaneousadipose tissue with the skin.

The entire area of skin was examined for any areas of necrosis anddamage. Large lesions were obvious, and small lesions were detectable asan area of yellowing tissue. The entire area of skin was examined, andlesions found related to the injection marks on the skin. Aftermeasurement with callipers and photography, samples of lesion tissuefrom each site, and control samples from sites distant from anyinjection site were removed and stored in formalin buffered saline forhistology.

The sizes of the lesions seen in at least 3 independent studies areshown in Table 2. Photographs are provided as FIGS. 5 (a) to (d). FIG. 5(d) shows the fat cells in a section from a control animal, where thefat cells have a large triglyceride droplet stored in the centre of thecell (which appears white in the photographs). Contrast FIG. 5 (a), (b)and (c) which are typical of the animals treated according to theinvention with antisera to antigens A, B and C, respectively, in whichthe dark areas represent lymphocyte cells infiltrating the adiposetissue.

                  TABLE 2                                                         ______________________________________                                        Effects of antisera raised against individual specific antigens               on adipose tissue in vivo                                                     Antiserum to:                                                                             Dose (ml) Area of destruction (mm)                                ______________________________________                                        Whole membrane                                                                            2         35                                                                  0.4       6.5                                                                 0.2       3.5                                                                 0.02      0                                                       Antigen                                                                       A           2         2                                                       B           2         3                                                       C           2         3.5                                                     D           2         2                                                       E           2         0                                                       H           2         0                                                       1           2         0                                                       2           2         0                                                       3           0                                                                 4           2         0                                                       5           2         0                                                       Non-immune  2         0                                                       serum control                                                                 ______________________________________                                    

Pigs received a single subcutaneous injection of antiserum at varioussites on the back, After 7 days the size of the site of adipocytedestruction was determined.

Results are the mean values obtained from at least 3 separateexperiments.

A problem in evaluating the results of Table 2 concerns the effectiveamounts of antibody raised. The electroelution procedure employed inpreparation of the specific antigens has probably resulted in somedegradation of proteins. Therefore, the antisera might have been raisedagainst components of the higher relative molecular mass antigens,rather than full length proteins. This makes it particularly difficultto compare amounts of effective epitope even with the benefit of theELISA data.

Notwithstanding these limitations, it was surprising to note that someantisera (anti-A and -B) which reacted strongly with adipocyte plasmamembranes In ELISA also elicited adipocyte lysis in vivo whilst all theothers except C and D, several of which reacted at least as well InELISA, failed to produce adipocyte destruction (compare FIG. 3 and Table2).

We claim:
 1. An isolated antigen present in the plasma membrane ofmature porcine white adipocytes, which is not detectable in porcineliver, kidney, spleen, brain, cardiac muscle, skeletal muscle or lung orin porcine erythrocytes, which reacts with antisera raised against saidadipocytes, said antigen being selected from the group consisting of a37, 50, 51, and 121 KiloDaltons relative molecular mass antigen asdetermined by SDS-PAGE using markers of relative molecular mass 29, 45,66, 97, 116, and 205 KiloDaltons.
 2. Isolated antibodies that arespecific to one of said antigens claimed in claim
 1. 3. Isolatedantibodies according to claim 2 which are monoclonal.
 4. A method ofreducing fat in pigs by active immunization which comprisesadministering to the pig an amount of an immunogen which comprises anantigen claimed in claim 1, said immunogen having been renderedeffective to elicit an immune response to adipocytes.
 5. A methodaccording to claim 4 wherein the immunogen is rendered effective toelicit an immune response by conjugation to a carrier protein.
 6. Amethod of reducing fat in pigs by passive immunisation which comprisesadministering antibodies claimed in claim 2 or 3 to the pig in an amounteffective to immunise the pig against fat deposition.